Basic Confocal Microscopy.pdf

Basic Confocal Microscopy

UNIT 14.11

Confocal microscopy produces sharp im-

ages of structures within relatively thick speci-

mens (up to several hundred microns). It is

particularly useful for examining fluorescent

specimens. Thick fluorescent specimens

viewed with a conventional widefield fluores-

cent microscope appear blurry and lack contrast

because fluorophores throughout the entire

depth of the specimen are illuminated and fluo-

rescence signals are collected not only from the

plane of focus but also from areas above and

below. Confocal microscopes selectively col-

lect light from thin (

THE BASIS OF OPTICAL

SECTIONING

Confocal microscopes accomplish optical

sectioning by scanning the specimen with a

focused beam of light and collecting the fluo-

rescence signal from each spot via a spatial

filter (generally a pinhole aperture) that blocks

signals from out-of-focus areas of the speci-

men. The physical basis of optical sectioning

in fluorescence confocal microscopy is illus-

trated in Figure 14.11.2. A point light source

(typically a laser) evenly illuminates the back

focal plane of the objective, which focuses the

light to a diffraction-limited spot in the speci-

men. The irradiation is most intense at the focal

spot, although areas of the specimen above and

below the focal spot also are illuminated. Fluo-

rescent molecules excited by the incident light

emit fluorescence in all directions. The fluores-

cence collected by the objective comes to focus

in the image plane, which is conjugate (confo-

cal) with the focal plane in the specimen. A

pinhole aperture in the image plane allows

fluorescence from the illuminated spot in the

specimen to pass to the detector but blocks light

from out-of-focus areas.

The diameter of the pinhole determines how

much of the fluorescence emitted by the illu-

minated spot in the specimen is detected, and

the thickness of the optical section. From wave

optics we know that a point light source in the

plane of focus of an objective produces a three-

dimensional diffraction pattern in the image

plane. The cross section at the image plane is

an Airy disk (see Fig. 14.10.9), a circular dif-

fraction pattern with a bright central region.

The radius of the bright central region of the

Airy disk in the reference frame of the specimen

is given by

R Airy = 0.61

m) optical sections

representing single focal planes within the

specimen. Structures within the focal plane

appear more sharply defined than they would

with a conventional microscope because there

is essentially no flare of light from out-of-focus

areas. A three-dimensional view of the speci-

men can be reconstructed from a series of

optical sections at different depths.

The confocal microscope is the instrument

of choice for examining fluorescence-stained

cells in tissue slices or small, intact organisms

such as Drosophila (Fig. 14.11.1A,B) and ze-

brafish embryos. It is also useful for localizing

fluorescent-tagged molecules in dissociated

cells (Fig. 14.11.1C,D). Its sensitivity even al-

lows fluorescence in living specimens to be

monitored, making it feasible to follow the

movements in living cells of fluorescent probes

such as the green fluorescent protein (GFP; Fig.

14.11.1D). In addition, some types of confocal

microscopes can be configured to perform pho-

tobleach experiments (Fig. 14.11.1D) and to

photoactivate “caged” molecules (molecules

that are inactive until released with UV illumi-

nation).

Biologists use confocal microscopy in a

number of creative ways that are beyond the

scope of this article. The information presented

herein is intended to provide background and

practical tips needed to get started with confo-

cal microscopy. An excellent source of theoreti-

cal and technical information is the Handbook

of Biological Confocal Microscopy (1995; ed-

ited by J. Pawley). Also recommended are Cell

Biological Applications of Confocal Micros-

copy (1993; edited by B. Matsumoto), a good

source of practical information; Confocal Mi-

croscopy (1990; edited by T. Wilson), for theo-

retical background; and Video Microscopy

(1997; Inoue and Spring) for fundamentals of

microscopy.

∼

/NA

where

is the emission wavelength and NA is

the numerical aperture of the objective (see UNIT

14.10 for a discussion of NA). At the image plane

(the location of the pinhole aperture), the radius

of the central region is R Airy multiplied by the

magnification at that plane (for a more com-

plete explanation see Wilson, 1995).

Adjustment of the pinhole to a diameter

slightly less than the diameter of the central

region of the Airy disk allows most of the light

from the focal point to reach the detector and

reduces the background from out-of-focus ar-

eas by

In situ

Hybridization

and Immuno-

histochemistry

∼

1000-fold relative to widefield micros-

Contributed by Carolyn L. Smith

Current Protocols in Molecular Biology (1998) 14.11.1-14.11.12

14.11.1

Supplement 44

Figure 14.11.1 Applications of laser scanning microscopy. ( A,B ) (From W. Oldenwald; see

Kamabadur et al., 1998.) 3-D analysis of thick specimens. Different neuronal populations of an

∼ 250- µ m-thick Drosophila embryo were immunolabeled with antibodies against three transcription

factors. A,

, 0.8-NA objective using a detector pinhole

diameter of ∼ 1.3 Airy units. Labeled neurons in the plane of focus appear sharply defined, while those

outside it are not visualized. B, projection (superimposition) of 65 optical sections collected at 2- µ m

intervals in the z axis. Neurons at different focal planes appear to overlap in this flattened image, but

are distinct in a 3-D reconstruction. ( C ) Localization of intracellular structures. Dissociated rat

fibroblasts were immunolabeled with anti-tubulin antibodies to visualize microtubules (green) and

stained with fluorescent probes for mitochondria (Mitotracker, red) and DNA (DAPI, blue). The image

is a projection of 20 optical sections collected at 0.3- µ m intervals in the z axis with 100 × , 1.4-NA

objective. ( D ) Measuring molecular motility. In a living fibroblast expressing a Golgi membrane protein

(galactosyltransferase) fused to GFP (S65T-GFP), GFP fluorescence (green) localized to the Golgi

complex, shown superimposed on a DIC image of the cell. After the first image was collected, the

boxed region (yellow) was scanned with full laser power; this photobleached the GFP in the boxed

area as shown in the second image collected

2.5-

m optical section collected with 25

∼

2 sec later. The rate of fluorescence recovery into the

photobleached zone (not illustrated) indicated that GFP-galactosyltransferase fusion is highly mobile

in Golgi membranes. This black and white facsimile of the figure is intended only as a placeholder;

for full-color version of figure go to http://www.currentprotocols.com/colorfigures

∼

Basic Confocal

Microscopy

14.11.2

Supplement 44

Current Protocols in Molecular Biology

copy (Sandison et al., 1995). The separation of

the in-focus signal from the out-of-focus back-

ground achieved by a properly adjusted pinhole

is the principle advantage of confocal micros-

copy for examination of thick specimens (see

Fig. 14.11.1A,B).

Point illumination and the presence of a

pinhole in the detection light path also produces

improved lateral and axial resolution relative to

conventional microscopy (Table 14.11.1). The

actual extent of improvement depends on the

size of the pinhole. Near-maximal axial resolu-

tion is obtained with a pinhole radius

TYPES OF CONFOCAL

MICROSCOPES

Several types of confocal microscopes are

available, each having unique features and ad-

vantages. The types most commonly used for

examining fluorescence specimens are laser-

scanning confocal microscopes. These micro-

scopes, as their name implies, use lasers as light

sources and collect images by scanning the

laser beam across the specimen.

Lasers provide intense illumination within

a narrow range of wavelengths. The emission

wavelengths of several types of lasers, together

with the excitation spectra of familiar fluoro-

phores, are illustrated in Figure 14.11.3. Mixed

krypton-argon gas lasers are popular for multi-

wavelength confocal microscopy because they

emit at three well-separated wavelengths (488,

568, and 647 nm) that can be used to simulta-

neously image two or three fluorophores (e.g.,

FITC, lissamine rhodamine, and Cy5). The

disadvantage of krypton-argon lasers is that

R Airy whereas optimal lateral resolution is ob-

tained with a pinhole less than 0.3

∼

0.7

R Airy

(Wilson, 1995). However, a pinhole smaller

than

R Airy significantly reduces the total

signal, a sacrifice that may not be worth the gain

in resolution, especially when imaging dim

samples. In fluorescence imaging, resolution

also is influenced by the emission and excita-

tion wavelengths (Table 14.11.1).

∼

0.7

photodetector

confocal pinhole

dichroic mirror

point

light

source

objective lens

specimen

focal plane

Figure 14.11.2 The basis of optical sectioning in confocal epifluorescence microscopy. Illumina-

tion from the point light source is reflected by the dichroic mirror and focused by the objective lens

to a diffraction-limited spot within the specimen. Fluorophores within the focal spot as well as in the

cone of light above and below it are excited, emitting fluorescence at a longer wavelength than the

incident light. The fluorescence captured by the objective passes through the dichroic mirror

because of its longer wavelength. The confocal pinhole allows fluorescence from the plane of focus

in the specimen to reach the photodetector but blocks fluorescence from areas above and below

the plane of focus. Redrawn from Shotton (1993).

In situ

Hybridization

and Immuno-

histochemistry

14.11.3

Current Protocols in Molecular Biology

Supplement 44

Table 14.11.1 Theoretical Resolutions of Confocal and Conventional Microscopes a

Objective

λ ex /

λ em

, 0.4 NA, air

, 0.85 NA, air

, 1.4 NA, oil

Lat. res.

Ax. res.

Lat. res.

Ax. res.

Lat. res.

Ax. res.

Confocal fluorescence microscope

488/518

0.55

4.50

0.26

0.99

0.16

0.56

568/590

0.64

5.17

0.30

1.09

0.18

0.64

647/677

0.72

5.88

0.34

1.28

0.21

0.72

Conventional fluorescence microscope

518

0.79

6.48

0.37

1.43

0.24

0.93

590

0.90

7.38

0.42

1.63

0.28

1.06

680

1.04

8.50

0.49

1.88

0.32

1.22

a Data reprinted from Brelje et al. (1993) by permission of Academic Press.

λ ex and

λ em , excitation and emission

wavelengths; lat. res. and ax. res., lateral and axial resolutions.

Cascade blue

FITC TRITC LRSC

Cy5.18

100

300

350

400

450

500

550

600

650

700

Wavelength (nm)

argon

488 514

UV argon

351, 364

457

488 514 528

krypton-argon

488

568

647

krypton

478/482

520

568

647

676

helium-neon

543

595 633

helium-cadmium

325

354

422

Figure 14.11.3 Comparison of the emission wavelengths of various lasers and the excitation

spectra of representative fluorophores. The lasers most commonly used for laser-scanning confocal

microscopy are air-cooled argon (488 and 514 nm), krypton-argon, and helium-neon lasers. UV

argon lasers generally require water cooling and are more expensive. They may be configured to

provide only UV wavelengths (351 nm and 364 nm) or both UV and longer wavelengths. Data for

the excitation spectra of Cascade blue, fluorescein (FITC), tetramethylrhodamine (TRITC), lis-

samine rhodamine (LRSC), and cyanine 5.18 (Cy5.18) are from Wessendorf and Brelje (1993) and

were downloaded from the web page of Aryeh Weiss, http://optics.jct.ac.il/ ∼ aryeh/Spectra. Modified

from Brelje et al. (1993).

Basic Confocal

Microscopy

14.11.4

Supplement 44

Current Protocols in Molecular Biology

their life spans are short (

2000 hr). Another

way to achieve multiwavelength excitation is

to combine the outputs of two or more lasers.

Several methods have been devised for scan-

ning the sample with the laser beam to illumi-

nate different positions in the specimen. The

most common method employs a pair of galva-

nometer mirrors to both scan the laser beam

across the specimen and collect the fluores-

cence emitted from the specimen (Fig.

14.11.4). One galvanometer mirror scans se-

quential spots along the x axis, and the second

mirror moves from line to line in the y axis. The

fluorescence emission is separated from the

illuminating beam by a dichroic beam splitter

and is directed to a photomultiplier tube which

collects the fluorescence produced as each spot

in the specimen is illuminated. The photodetec-

tor output is converted to a digital image that

can be displayed on a monitor and stored as a

digital image file for later analysis. Most laser-

scanning confocal microscopes have 8-bit digi-

∼

tizers that encode 256 gray levels, although

some recent models have 12- or 16-bit digitiz-

ers. Collection of a full-size image (typically

1024

2 sec. Laser-scan-

ning microscopes that employ galvanometer

mirror scanners sometimes are called “slow-

scan” microscopes because of their relatively

slow image acquisition rates. Slow-scan micro-

scopes are available from several sources (Bio-

Rad, Zeiss, Leica, Olympus, Nikon, Molecular

Dynamics, and Meridian; see APPENDIX 4 ).

The movements of the galvanometer mirrors

in laser-scanning microscopes are under the

control of a computer, providing flexibility in

the scanning pattern. For example, it is possible

to “zoom” a region of interest (visualize it at

higher magnification) by reducing the scan area

and the distances between sample points. In

addition, many laser-scanning microscopes

have the ability to repetitively scan a single line

or to “park” the scanner to monitor fluores-

cence at a single spot. The latter technique is

1024 pixels) takes

∼

krypton-argon

laser

variable

pinhole

emission

filter

specimen

line-selection filter

objective

photomultiplier

tube 2

neutral-density filter

mirror

dichroic

beam

splitter 1

dichroic

beam

splitter 2

photomultiplier

tube 1

microscope

scanner

488-nm laser beam

lissamine rhodamine

FITC

568-nm laser beam

Figure 14.11.4 The light path of a laser-scanning confocal microscope set up for simultaneous

imaging of FITC and lissamine rhodamine. The 488-nm and 568-nm lines of a krypton-argon laser

are reflected by dichroic beam splitter 1 into the optical axis of the microscope. The scanner contains

two galvanometer mirrors, which generate the x and y axis movements of the beam. The beam is

reflected by a mirror into the objective which focuses the beam onto the specimen. The specimen

is scanned line by line in a raster pattern. Fluorescence emitted by the specimen as each spot is

illuminated travels the reverse path through the scanning system. The FITC fluorescence (peak at

520 nm) and lissamine rhodamine fluorescence (peak at 590 nm) pass through dichroic beam

splitter 1 to dichroic beam splitter 2, which transmits the lissamine rhodamine fluorescence to

photomultiplier tube 1 and reflects the FITC fluorescence to photomultiplier tube 2. A variable

pinhole in front of each photodetector blocks light from out-of-focus areas of the specimen while

allowing light from the illuminated spot to reach the detector.

In situ

Hybridization

and Immuno-

histochemistry

14.11.5

Current Protocols in Molecular Biology

Supplement 44

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